The long-term objective of this research is to use cell biological and biochemical techniques to understand the molecular nature of the kinetochore-microtubule (MT) interface in vertebrate cells. For chromosomes to properly segregate during mitosis, they must firmly attach to dynamically growing and shortening MT plus- ends. They do so via the kinetochore, a large protein assemblage built at the primary constriction of mitotic chromosomes. This linkage is likely complicated, as it must be robust to resist the forces of chromosome bi- orientation, yet flexible to allow for fluid growth and shortening of bound MT plus-ends. Kinetochores must also regulate the strength of this attachment, since incorrectly attached MTs must be released, and those that are correctly attached must be stabilized. The kinetochore-associated NDC80 complex is required for generating stable kinetochore-MT attachments in eukaryotic cells, but how this complex builds and regulates binding sites for the plus-ends of spindle MTs remains one of the most important unanswered questions in the mitosis field. This proposal is designed to answer the following questions: What domains of the NDC80 complex make up the direct points of contact with MT plus-ends? Are the complexes tethered together in a sleeve for the MT plus-ends to insert into? If so, how are the complexes tethered together? Do weakly-associated NDC80 complexes diffuse along the MT lattice to facilitate chromosome congression? Or, alternatively, are the points of contact between kinetochores and MTs made up of high affinity binding interactions that require continual release and re-binding to drive chromosome movement? Is kinetochore-MT binding strength regulated through phosphorylation of the NDC80 complex by Aurora B kinase? What phosphatase counter-acts the kinase activity to ensure kinetochore-MT stabilization? These questions will be answered using the following approaches: First, a gene silence/rescue strategy for NDC80 complex components will be developed in PtK1 cells to unambiguously assess kinetochore-MT attachment phenotypes in cells expressing mutant NDC80 complexes. Second, biochemical and biophysical experiments using NDC80 mutants will be carried out to understand mechanistically how NDC80 complexes bind to and translocate along MTs, and which features of the complex are responsible for physically coupling plus-end MT dynamics to force production for chromosome movement. Third, protein-protein interactions will be mapped at the kinetochore-MT interface for the first time in vivo through the development of kinetochore-specific fluorescence interaction assays. These studies will provide answers to a critical set of unresolved questions in the mitosis field, and the developed techniques will be applicable to further study of mitotic proteins and processes. Relevance: Progression through mitosis with incorrect kinetochore-MT attachments is a major cause of aneuploidy, which has been linked to the initiation and progression of human tumors and also to the formation of birth defects. Thus, understanding how cells generate and regulate kinetochore-MT attachments is of critical importance to human health.